This application is the National Stage of International Application No. PCT/IT97/00163, filed Jul. 10, 1997.
The present invention relates to the field of neurology. Subject of the invention are mutants of the human ciliary neurotrophic factor (hCNTF) that show a range of action different from that of the wild-type molecule. These variants are characterised by the fact that they have a strongly reduced ability to interact with the LIF receptor (LIFR). As a consequence they have an extremely reduced biological activity on the majority of cells responding to CNTF. However, they retain a high biological activity on certain neurone cells, probably because the latter express large amounts of LIFR and/or CNTF receptor xcex1 (CNTFRxcex1) bound to the membrane. This property can be used to reduce the peripheral side effects resulting from administration of CNTF.
The human ciliary neurotrophic factor (hCNTF) is a neurocytokine of 23 kDa. It is expressed in Schwann cells and astrocytes, and exerts potent stimulatory effects on the survival and differentiation of a variety of neuronal and glial cells (both in vitro and in vivo), including motor neurons sensor neurons, sympathetic neurons, neurons of the hippocampus and oligodendrocytes (1, 2). The physiological role of CNTF would appear to be that of acting as a factor involved in the prevention of neuronal degeneration following injury (2). This protective action, which CNTF has shown in vivo on many nerve cells, has given hopes for its clinical application in the treatment of human neurodegenerative diseases (3).
As well as its neuroprotective action, however, it has been seen that CNTF also elicits in vivo a wide variety of effects, consisting of loss of weight and anorexia, acute-phase response and fever. There is therefore a problem with the pharmacological use of CNTF because of these side effects.
Like other growth factors, and in particular cytokines, CNTF exerts its actions through the binding, sequential assembly, and activation of a multisubunit receptor complex (4, 5). The receptor complex of which CNTF forms a part is made up of six subunits (similarly to the case of interleukin 6) (6). The complex is made up of two CNTF molecules, two xcex1-receptor specific molecules forming a low-affinity bond with CNTF (CNTFRxcex1) and two signal transducing subunits (LIFR and gp130) (11). The CNTF sites involved in binding with gp130and with the specific receptor can be identified by analogy to interleukin 6 (7, 8, 10). The bonding site for CNTF and the LIF receptor (LIFR) has not yet been identified.
Multiple alignment of human, rabbit, rat and mouse CNTF sequences with other cytokine sequences that bind the LIF receptor (such as LIF, oncostatin M and CT-1) reveals the presence of two conserved amino residues within the structural motif known as D1. The two amino acids are phenylalanine in position 152 and lysine in position 155. As will be shown in greater detail in the following examples, the two amino acids form part of the LIFR bonding site. On the basis of this notion, CNTF mutants were generated in which the two amino acids were replaced by alanine, and the sequences of both the wild-type molecule (SEQ ID NO:1) and the mutants which are the object of the present invention are given in table 1 (for the sake of simplicity the whole amino acid sequence is not given, only the amino acid positions from 152 to 167). Although the literature indicates that one of the two mutants (Lys155Ala/CNTF) is completely inactive on hen neurons (10), the biological activity of both mutants was evaluated, and it was demonstrated that, contrary to previous reports (10), they act as powerful agonists on the membrane receptor of certain neuronal cells, whereas their biological activity is strongly reduced when they are bound to the LIF receptor or to the soluble CNTF receptor. A possible interpretation of this phenomenon may be the following: as the biological effects of these molecules depend on the concentrations of CNTFRxcex1 and LIF receptor, they are amplified when the local receptor concentration is very high, as is the case in receptors that are bound to the cell membrane of certain neuronal cell populations.
The discovery that these molecules exert a biological activity on neuronal cells expressing membrane-bound receptors and that on the contrary they show an extremely weak biological activity when interacting with the soluble receptor indicates that molecules that are modified in the positions indicated can be used for specific reaction with neuronal cells without causing peripheral side effects mediated by the soluble receptor bond.
Subject of the present invention are therefore variants of the human ciliary neurotrophic factor (hCNTF), characterised by replacement of the phenylalanine 152 and/or of the lysine 155 with alanine and comprising in particular an amino acid sequence chosen from the sequences indicated in SEQ ID NO:3 to SEQ ID NO:7.
The molecules represented by SEQ ID NO: 3 to SEQ ID NO:6 have a reduced ability to bond to the LIF receptor and to activate it by means of soluble CNTFRxcex1, and on the contrary act as agonists on the CNTFR of certain neuronal cells. The molecule represented by SEQ ID NO:7 acts as a competitive antagonist for the CNTFRxcex1 receptor bond.
The present invention also extends to pharmaceutical preparations making use of these molecules for treatment of diseases or pathologies of the nervous system, or of other pathologies involving cells that respond to CNTF, including diseases or pathologies of the nervous system that cause damage to the nervous system itself.
Table 2 shows the data relating to CNTFRxcex1 receptor binding activities of the various mutants of CNTF. The concentration of protein needed to inhibit by 50% (IC50) the binding of biotinylated CNTF or biotinylated MUT-DH to immobilised CNTFRxcex1 was determined. Relative binding is the ratio (IC50 of CNTF)/(IC50 of tested protein).